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. 2024 Oct 23;9(11):e00647-24. doi: 10.1128/msphere.00647-24

A whole-cell pertussis vaccine engineered to elicit reduced reactogenicity protects baboons against pertussis challenge

Parul Kapil 1,#, Yihui Wang 1,#, Kelsey Gregg 1, Lindsey Zimmerman 1, Damaris Molano 1, Jonatan Maldonado Villeda 1, Peter Sebo 2, Tod J Merkel 1,✉
Editor: Marcela F Pasetti3
PMCID: PMC11580402  PMID: 39441011

ABSTRACT

Whole-cell pertussis (wP) vaccines introduced in the 1940s led to a dramatic reduction of pertussis incidence and are still widely used in low- and middle-income countries (LMICs) worldwide. The reactogenicity of wP vaccines resulted in reduced public acceptance, which drove the development and introduction of acellular pertussis (aP) vaccines in high-income countries in the 1990s. Increased incidence of pertussis disease has been observed in high-income countries following the introduction of aP vaccines despite near universal rates of pediatric vaccination. These increases are attributed to the reduced protection against colonization, carriage, and transmission as well as reduced duration of immunity conferred by aP vaccines relative to the wP vaccines they replaced. A reduced reactogenicity whole-cell pertussis (RRwP) vaccine was recently developed with the goal of achieving the same protection as conferred by wP vaccination but with an improved safety profile, which may benefit countries in which wP vaccines are still in routine use. In this study, we tested the RRwP vaccine in a baboon model of pertussis infection. We found that the RRwP vaccine induced comparable cellular and humoral immune responses and comparable protection following challenge relative to the wP vaccine, while significantly reducing injection-site reactogenicity.

IMPORTANCE

The World Health Organization (WHO) recommended in 2015 that countries administering wP vaccines in their national vaccine programs should continue to do so, and that switching to aP vaccines for primary infant immunization should only be considered if periodic booster vaccinations and/or maternal immunization could be assured and sustained in their national immunization schedules (WHO, Vaccine 34:1423–1425, 2016, https://doi.org/10.1016/j.vaccine.2015.10.136). Due to the considerably higher cost of aP vaccines and the larger number of doses required, most LMICs continue to use wP vaccines. The development and introduction of a wP vaccine that induces fewer adverse events without sacrificing protection would significantly benefit countries in which wP vaccines are still in routine use. The results of this study indicate this desirable goal may be achievable.

KEYWORDS: whole cell vaccines, pertussis, baboon model

INTRODUCTION

Introduction of the whole-cell pertussis vaccine (wP) in the second half of the 20th century led to a dramatic reduction of pertussis incidence (1) (CDC; http://www.cdc.gov/pertussis/surv-reporting.html). Although highly effective, vaccination with the early versions of the wP vaccines often resulted in mild adverse reactions, including fever, loss of appetite, persistent crying, drowsiness, vomiting, fretfulness, local redness, and induration at the site of immunization. Rarely, it resulted in more severe adverse reactions, including high fever, febrile seizures, and hypotonic hyporesponsive episodes (2–4). In response to reduced public acceptance of the wP vaccine driven by the high rates of reactogenicity and perceived risk, acellular pertussis (aP) vaccines were developed and introduced in high-income countries (5). Although the wP vaccine was replaced by acellular pertussis (aP) vaccines in high-income countries, wP is still the primary vaccine used in most low- and middle-income countries (6, 7), accounting for approximately 70% of pertussis vaccinations delivered to infants in the first year of life worldwide (Table S1).

The aP vaccine is highly effective in controlling disease and preventing infant mortality from pertussis (8–11). However, pertussis cases have increased in countries following the introduction of aP vaccines (12, 13). Pertussis outbreaks in Australia between 2009 and 2011 peaked with a disease incidence of 125 per 100,000 people (14, 15). Increasing incidence of pertussis was recorded across the United States of America, with peaks in 2010, 2012, and 2014 (16, 17), and in parts of the European Union in 2012 and 2024 (18–20).

Early work by Mills and Readhead observed a T helper type 1 (Th1) or T helper type 2 (Th2) polarization of immune responses after immunization with either wP or aP vaccines, respectively (21). Using a baboon model of pertussis, Warfel et al. revealed that although aP-vaccinated animals are protected from disease and exhibit no outward signs of infection, they fail to clear bacteria from the airway and remain colonized longer than unvaccinated controls (22). Additionally, aP-vaccinated baboons transmitted pertussis to unvaccinated cage-mates (22). Warfel et al. also demonstrated in the baboon model that aP vaccines prime a Th2-dominated T-cell response, whereas wP vaccines drive Th1/Th17 T-cell responses (23–25). Recent work by several groups using the mouse model of airway infection by Bordetella pertussis showed that clearance of B. pertussis from the airway requires IL-17 and IFN-γ-secreting tissue-resident memory T cells (TRM) that play a key role in early protection of the airway mucosa from B. pertussis infection. These immune responses are induced by natural infection and wP vaccination but not by aP vaccination (22, 25–31) and were recently confirmed in humans (32). The observation that aP vaccines do not prevent colonization, carriage, and transmission, in contrast to the wP vaccines they replaced, potentially explains the resurgence of pertussis in aP-vaccinated populations (33, 34).

Despite their reactogenicity, the wP vaccines are still broadly used by many countries to effectively control pertussis. To improve upon the current wP vaccines, a reduced-reactogenicity whole cell (RRwP) pertussis vaccine was developed by the Sebo group as a safer alternative (K. Škopová, J. Holubová, B. Bočková, E. Slivenecká, et al., submitted for publication). The vaccine was generated on the background of a low-passage stock of the Fim2 (VS67) and Fim3 (VS377) serotype B. pertussis strains. The two strains were genetically modified, eliminating the function of three genes. The BP0398 gene was removed (ΔlgmB), deleting the glucosyl transferase responsible for adding inflammatory glucosamine modifications to the terminal phosphate groups of B. pertussis lipid A (35–37). The lipid A from this genetically modified pertussis strain exhibits significantly reduced TLR4 activation and endotoxic potency (36). The gene encoding dermonecrotic toxin (DNT) was also removed (38, 39). Finally, pertussis toxin was genetically detoxified by replacing key residues in the toxin’s S1 subunit. The arginine at position 9 of the processed S1 was substituted with lysine (R9K), and the glutamic acid at position 129 was substituted with glycine (E129G), eliminating the ADP-ribosylating enzyme activity with minimal impact on the original structure (40–44). Formulation of the two strains into whole-cell vaccine results in a vaccine that is FIM2/3, ΔlgmB, DNTNEG, and PTR9K/E129G. In this study, the baboon model was used to evaluate the ability of the RRwP vaccine to protect against disease and reduce carriage relative to a standard wP vaccine. Our results indicate the RRwP vaccine is less reactogenic and retains the ability to prevent disease and reduce colonization.

RESULTS

RRwP vaccine exhibits less reactogenicity at the injection sites than wP vaccine

To determine if the RRwP vaccine was less reactogenic than the comparable wP vaccine when injected intramuscularly in baboons, while retaining the ability to protect against disease and reduce bacterial carriage, we vaccinated four baboons per group at 2, 4, and 6 months of age with the human dose of wP or aP vaccine as indicated on the label and the equivalent dose of RRwP. The injection sites were evaluated on days 1, 3, and 5 following each vaccination by a veterinarian who was blinded to the vaccination groups. No signs of redness, induration, or swelling were observed in any of the animals (data not shown). To closely monitor temperature through the vaccination series, the animals were implanted with microCT temperature dataloggers (Star-Oddi LTD, Gardabaer, Iceland). The dataloggers were recovered from the animals at the end of the study, and the data were downloaded and analyzed. There was no significant difference in systemic body temperature following vaccination between the three vaccine groups (Fig. 1).

Fig 1.

The point plot shows the difference in the area under the curve for systemic body temperature post-vaccination with aP, wP, and RRwP. The data points indicate that there was no significant difference in the changes of body temperature between the groups.

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Difference in systemic body temperature post-vaccination. The area under the curve (AUC) of the body temperature recorded by the dataloggers was calculated for the same 18-h period during the 3 days before each vaccination as described in Materials and Methods, and the average pre-vaccination body temperature AUC was calculated and subtracted from the body temperature AUC of the same animal on day 1 post-vaccination recorded for the same 18-h period. The 18-h interval between 3:00 PM and 9:00 AM was chosen to avoid the impact of sedation on body temperature. Data are presented as mean ± standard deviation (SD). A repeated measures two-way ANOVA did not detect any significant differences between groups.

Previous studies have shown that baboons and macaques are less sensitive to lipopolysaccharide (LPS) than humans (45, 46). To observe injection site reactogenicity following vaccination, we vaccinated four baboons with high-dose wP vaccine and four baboons with high-dose RRwP vaccine. These vaccines were prepared by concentrating four doses of vaccine to the volume of a single dose. Following injection, the injection sites were examined on days 1, 2, 3, and 5 by a veterinarian blinded to treatment, and relative scores were assigned for redness, induration, and swelling. In addition, the surface temperature was measured at the injection site, and the rectal body temperature was recorded. Peak reactogenicity was observed approximately 24 h post-injection. Both the high-dose wP- and high-dose RRwP-vaccinated baboons exhibited some degree of injection site reactogenicity, although injection site reactions were more evident in wP-vaccinated baboons (Fig. 2). The injection site reactions were significantly reduced at 48 h and fully resolved by day 5 (data not shown). Significantly higher scores in redness and slightly higher scores in induration were recorded for the wP-vaccinated baboons relative to the RRwP-vaccinated baboons (Fig. 2). None of the RRwP-vaccinated baboons exhibited redness at the injection site. In contrast, all four wP-vaccinated baboons exhibited redness and induration (Fig. 2). The differences in injection site induration and swelling between the wP- and RRwP-vaccinated animals did not reach statistical-significance, but one of the four wP-vaccinated baboons had severe induration, and two had severe swelling (clinical score of 3). Only two of the RRwP-vaccinated animals exhibited mild induration, and one had mild swelling with a clinical score of 1 (Fig. 2). There were no significant differences in the injection site skin temperature or body temperatures measured rectally between the RRwP- and wP-vaccinated animals (Fig. 2). The eight high-dose vaccinated baboons were not evaluated further.

Fig 2.

The point plots compare wP and RRwP across injection site redness, induration, swelling, temperature, and core temperature with wP trends higher than RRwP in general. Statistical significance is noted between wP and RRwP for redness.

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Injection site reactions measured on day 1 post-injection of high-dose RRwP and high-dose wP vaccines. Four baboons each were vaccinated with high-dose wP or high-dose RRwP vaccines prepared as described in Materials and Methods. On day 1 post-vaccination, the injection sites of all eight animals were evaluated for redness, induration, swelling, and temperature by a veterinarian blinded to treatment groups. Relative clinical scores were assigned by the veterinarian. The non-injected arm served as a negative control for each animal. The injection site temperature is reported as a ratio of the injected arm injection site temperature relative to the control arm mock injection site temperature. In addition to the evaluation of the injection site, the core body temperature was measured and reported as a ratio relative to the temperature of each animal measured on day 0 just before vaccination. Comparisons between groups were made using the nonparametric two-tailed unpaired Mann–Whitney U test. Data are presented as mean ± standard deviation (SD). Statistical difference in site-reactions between high-dose vaccines was observed for redness.

RRwP vaccine induced comparable cellular and humoral immune responses compared with wP vaccine post-vaccination

Two months following the third vaccination, whole blood was collected from the unvaccinated, aP-vaccinated, wP-vaccinated, and RRwP-vaccinated baboons. Peripheral blood mononuclear cells (PBMC) were isolated and stimulated with heat-killed B. pertussis. Enzyme-linked immunosorbent assays (ELISAs) were performed with the cell supernatants collected after 72 h of stimulation to determine the levels of cytokines indicative of Th1 (IFNγ), Th2 (IL5), and Th17 (IL17a) T-cell responses. As reported in previous studies, aP vaccination induced relatively high levels of IL5 production, but no statistically significant increase in IFNγ or IL17a was observed (Fig. 3). In contrast, wP vaccination induced IFNγ and IL17a with no increase in IL5 (Fig. 3). With RRwP vaccination, no increase in IL-5 was observed (Fig. 3). Additionally, although the restimulated PBMCs isolated from the RRwP vaccinated group produced slightly less IFNγ, they produced comparable amount of IL17 to those isolated from the wP group (Fig. 3).

Fig 3.

The dot plots show cytokine productions for IFNγ, IL5, and IL17a of restimulated PBMCs from vaccinated and naive baboons. The plots indicate that wP and RRwP have higher IFNγ and IL17a levels than aP and Unv, whereas IL5 is higher in aP.

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T-cell responses of aP, wP, and RRwP-vaccinated baboons. PBMCs were isolated from whole blood collected from vaccinated baboons following the third vaccination and from age-matched unvaccinated baboons. The cells were stimulated with heat-killed B. pertussis, and supernatants were collected 72 h later and were assessed for (A) IFNγ, (B) IL5, and (C) IL17a protein levels using ELISA as described in Materials and Methods. Comparisons between groups were made using the nonparametric two-tailed unpaired Mann–Whitney U test. Data are presented as mean ± standard deviation (SD).

Two months after the last vaccination, anti-HKBp and anti-PT IgG titers were characterized with serum collected from the vaccinated animals. The RRwP vaccine induced comparable anti-HKBp IgG titers compared with wP vaccine, whereas the anti-PT IgG titers trended lower than the ones induced by wP and aP vaccinations (Fig. 4).

Fig 4.

The figure shows pertussis-specific serum antibody titers from baboons post-vaccinations with aP, wP, or RRwP. No significant difference were observed between wP and RRwP for anti-HKBp IgG titers, while anti-PT IgG titers trends lower in RRwP.

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Pertussis-specific serum IgG responses 2 months after aP, wP, or RRwP vaccination. Two months following the third vaccination, (A) anti-HKBp and (B) anti-PT IgG titers were measured with serum collected from the vaccinated animals using ELISA. Comparisons between groups were made using the nonparametric two-tailed unpaired Mann–Whitney U test. Data are presented as mean ± standard deviation (SD).

RRwP and wP vaccines exhibited comparable protection from B. pertussis challenge

The unvaccinated and vaccinated baboons were inoculated with B. pertussis strain D420 as described in Materials and Methods. The baboons were observed twice a day following the challenge to assess their overall health; coughing was observed but not quantified. Coughing fits were observed in all four unvaccinated animals between days 8 and 12. No coughing was observed in the aP, wP, or RRwP-vaccinated animals following challenge. Blood and nasopharyngeal washes were collected twice per week following challenge. A significant increase in white blood cells was observed in the unvaccinated baboons, peaking at over 30,000 cells/µL on day 7 post-challenge (Fig. 5), as is characteristic of the disease. In contrast, no increase in white blood cells was observed in the vaccinated animals post-challenge (Fig. 5).

Fig 5.

The line graphs compare CFU/mL and WBC/µL post-challenge for groups Unv, aP, wP, and RRwP, showing CFU decreasing with Unv starting higher, then declining, and WBC counts changing with Unv peaking around day 8 and other groups staying at baseline.

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Bacterial colonization and white blood cell count in vaccinated and unvaccinated baboons following B. pertussis challenge. Vaccinated and unvaccinated baboons (n = four per group) were inoculated with B. pertussis strain D420 as described in Materials and Methods. Blood and nasopharyngeal washes were collected twice weekly following inoculation. Nasopharyngeal washes were diluted and plated on Regan–Lowe plates, and the CFUs/mL of nasopharyngeal wash were enumerated following incubation. The numbers of circulating white blood cells/µL of whole blood were determined by complete blood cell differentiation as described in Materials and Methods.

Unvaccinated baboons were heavily colonized following challenge (Fig. 5). These animals remained colonized at the same high level for over 2 weeks and began to clear the infection between days 17 and 21. Bacterial numbers in the nasopharynx of these animals steadily declined between days 21 and the end of study on day 33. The aP-vaccinated animals were initially colonized at the same high level as the unvaccinated animals (Fig. 5). CFUs slowly dropped approximately 2 logs between days 3 and 17 and remained at approximately 104 CFUs/mL of nasopharyngeal wash until day 24. As observed in previous studies, colonization of the aP-vaccinated animals remained higher than that observed in the unvaccinated animals from day 24 until the study ended at day 33. Both the wP and RRwP animals were heavily colonized on day 3, and both groups steadily cleared the infection at the same rate over the following 30 days (Fig. 5).

RRwP vaccine induced comparable cellular and humoral immune responses compared with wP vaccine post challenge

On days 7, 14, and 21 post-challenge, PBMCs were isolated from all animals to characterize pertussis specific CD4 T-cell cytokine responses. After isolation, PBMCs were stimulated with HKBp and cultured in the presence of co-stimulatory anti-CD28 and anti-CD49d antibodies for 16 h before adding inhibitor for cellular protein transport as described in Materials and Methods. After staining for surface markers, the cells were fixed and permeabilized for intracellular staining with anti-IFNγ and anti-IL17a antibodies to capture cytokine production before acquisition on a BD LSRFortessa X-20 flow cytometer. Significant increase in IL17a-producing CD4 T cells and a slight increase in IFNγ-producing CD4 T cells was observed in unvaccinated animals at day 14 post-challenge, whereas aP-vaccinated animals showed no significant increase in either cytokine-producing CD4 T cells (Fig. 6). In comparison, RRwP- and wP-vaccinated animals exhibited higher percentages of IFNγ-producing CD4 T cells and IL17a-producing CD4 T cells as early as day 7 post-challenge. Unvaccinated animals exhibited percentages of IFNγ-producing CD4 T cells and IL17a-producing CD4 T cells comparable to RRwP- and wP-vaccinated animals but not until day 21 post-challenge (Fig. 6).

Fig 6.

The figure shows data points measuring IFNγ and IL17a producing CD4+ cell percentages over days 7, 14, and 21 post-challenge in groups Unv, aP, wP, and RRwP, indicating non-significant differences between wP and RRwP at different time points.

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Induction of IFNγ- and IL17-producing T cells in vaccinated and unvaccinated baboons following challenge. PBMCs isolated from animals on day 7, 14, and 21 post-challenge were subject to stimulation, staining, and flow cytometry analysis as described in Materials and Methods. Percentage of IFNγ- or IL17a-producing CD4 T cells were characterized.

To characterize the humoral responses following challenge, anti-HKBp and anti-PT IgG titers were characterized with serum collected before challenge and on day 7 and 28 post-challenge. Anti-HKBp and anti-PT IgG titers in RRwP-vaccinated animals were comparable to wP-vaccinated animals (Fig. 7). Animals vaccinated with aP vaccines showed similar anti-PT IgG titers but lower anti-HKBp IgG titers compared to both the wP and RRwP vaccines (Fig. 7).

Fig 7.

A scatter plot shows anti-HKBp and anti-PT serum IgG titers for groups Unv, aP, wP, and RRwP post-challenge at various time points. Plots show anti-HKBp titers in RU/mL and anti-PT titers in IU/mL, with non-significant differences between wP and RRwP.

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Pertussis-specific serum IgG responses to aP, wP, and RRwP vaccination pre- and post-challenge. Pre-challenge and on days 7 and 28 post-challenge, (A) anti-HKBp and (B) anti-PT IgG titers were measured with serum collected from the animals by ELISA. Data are presented as mean ± standard deviation (SD).

DISCUSSION

Although aP vaccines are highly effective in protecting against disease and are significantly less reactogenic than the wP vaccines they replaced, the enhanced protection conferred by wP vaccines against colonization, carriage, and transmission provides an important advantage for the control of pertussis in vaccinated populations. The World Health Organization’s Strategic Advisory Group of Experts on immunization (SAGE) recommended that national vaccine programs currently administering wP vaccination should continue to use wP vaccines for the primary vaccination series (7). SAGE cautioned that a switch from wP to aP vaccines for primary infant immunization should only be considered if the inclusion of periodic boosters and/or maternal immunization can be assured and sustained (7). For low- and middle-income countries, this has major financial implications due to the higher cost of aP vaccines and the larger number of doses required. The introduction of less-reactogenic wP vaccines would be beneficial for those countries continuing to use wP vaccines in their immunization programs.

In this paper, we utilized the baboon model to evaluate a wP vaccine produced using two B. pertussis strains engineered to have reduced reactogenicity and an improved safety profile through genetic modification of the B. pertussis lipooligosaccharide (LOS) biosynthetic pathway, genetic detoxification of PT, and deletion of the gene encoding dermonecrotic toxin (DNT). This RRwP vaccine was shown using the mouse model to be comparably immunogenic and protective relative to the isogenic wP vaccine (Škopová et al., submitted).

When baboons were vaccinated with the standard human dose of the wP and RRwP vaccines at 2, 4, and 6 months of age, we observed no significant difference in systemic body temperature or injection-site reactogenicity between all vaccination groups (Fig. 1). Endotoxin is the highly inflammatory portion of bacterial LPS and LOS and contributes considerably to the reactogenicity of wP vaccines (37, 47). Others have shown that wP vaccines with reduced endotoxic activity can be engineered either by modifying the manufacturing processes or by genetic manipulation of the LOS synthesis pathway of the vaccine strain (47, 48). The lack of observable reactogenicity for the standard wP vaccine in our studies could be the result of the efforts of manufacturers to reduce endotoxin levels in currently distributed wP vaccines through changes in the manufacturing process. Alternatively, it may be due to baboons having reduced sensitivity to LPS (45, 46). A previous study determined that a 1,000-fold higher concentration of bacterial LPS was required to elicit a similar systemic inflammatory response in baboons when compared to humans (46). This reduced sensitivity to LPS has also been observed in rhesus macaques (49). To see if a higher dose of wP vaccine could elicit reactogenicity in baboons and therefore be able to determine if the RRwP vaccine would have reduced reactogenicity, the baboons were vaccinated with concentrated wP and RRwP vaccines. The high-dose wP-vaccinated baboons had increased redness and induration at the injection site relative to the high-dose RRwP-vaccinated animals (Fig. 2). Although high-dose vaccine was required to observe reactogenicity in wP-vaccinated baboons, these results suggest the use of the RRwP vaccine, on a population scale, would result in a lower incidence of injection site reactions in humans relative to the wP vaccine.

PBMCs from RRwP-vaccinated baboons stimulated with heat-killed pertussis had a slightly reduced IFNγ response compared with wP-vaccinated baboons while showing comparable IL17a production (Fig. 3). Although IFNγ is not directly stimulated by lipid A in complex with TLR4, inflammatory responses, including IFNγ production are potentiated by the lipid A-TLR4 signaling complex (50, 51). Dampening these signaling pathways with the altered lipid A structure present in the RRwP vaccine may be contributing to a shift of the immune response, resulting in a reduced IFNγ response while having no apparent effect on the 1L17a response. After pertussis challenge, the animals vaccinated with wP and RRwP showed similar levels of initial colonization and rates of clearance, whereas aP-vaccinated animals exhibited prolonged colonization as reported in previous baboon studies (22). All vaccinated animals were protected from leukocytosis (Fig. 5). In contrast to the restimulated PBMCs isolated pre-challenge (Fig. 3), PBMCs isolated on days 7, 14, and 21 post-challenge showed similar percentages of IFNγ- and IL17a-producing CD4 T cells between wP- and RRwP-vaccinated animals (Fig. 6). This suggests that although RRwP vaccination does not induce production of IFN γ cytokine to the same extent as observed following wP vaccination, it does not impair the capacity of CD4 T cell to expand and produce IFN γ after B. pertussis infection. Two months after the last vaccination, animals vaccinated with wP or RRwP vaccines showed similar anti-pertussis whole-cell serum IgG titers, whereas RRwP vaccination elicited lower anti-PT serum IgG ELISA titers (Fig. 4). Similarly, lower anti-PT serum IgG titers were seen in RRwP-vaccinated animals compared with the wP-vaccinated animals at 5 months after vaccination and right before challenge. No significant differences were observed in anti-HKBp or anti-PT serum IgG titers between the two wP vaccines following infection (Fig. 7), and the in vivo anti-PT response induced by RRwP vaccination was effective. No leukocytosis, a PT-specific pathology (52, 53), was observed following challenge in RRwP-vaccinated animals (Fig. 5). We do n0t understand why anti-PT serum IgG titers were lower in RRwP-vaccinated animals relative to wP-vaccinated animals, and this response was observed regardless of whether wild-type PT or genetically inactivated PT was used as the ELISA plate-coating antigen (data not shown). Nevertheless, vaccination with the RRwP vaccine induced equally effective immune responses following infection relative to the wP vaccine, including anti-PT IgG titers.

In conclusion, our results indicate that relative to a comparable wP vaccine, the RRwP vaccine was less reactogenic but retained the ability to prevent disease and reduce colonization. The development and introduction of a wP vaccine that induces fewer adverse events without sacrificing protection could benefit countries in which wP vaccines are still in routine use. Clinical immunogenicity studies are needed to determine if these safety benefits are observed in humans.

MATERIALS AND METHODS

Bacterial strains and media

B. pertussis strain D420 was grown on Bordet–Gengou agar plates prepared with Bordet–Gengou agar (Becton Dickinson, Sparks, MD) containing 1% proteose peptone (Becton Dickinson, Sparks, MD) and 15% defibrinated sheep blood. Regan–Lowe plates were prepared from Regan–Lowe charcoal agar base (Becton Dickinson) with 10% defibrinated sheep blood (Quad Five, Ryegate, MT) and 40 µg/mL cephalexin.

Vaccination

Twelve baboons were vaccinated at 2, 4, and 6 months of age. Four were vaccinated with the full human dose of the commercial aP vaccine, Daptacel (Sanofi Pasteur Limited, Toronto, Canada), four were vaccinated with the full human dose of the commercial wP vaccine, Pentavac SD (Serum Institute of India, Pune, India), and four baboons were vaccinated with an experimental pentavaccine containing the RRwP component. Production of the RRwP vaccine was performed by Serum Institute of India under contract to the Institute of Microbiology of the Czech Academy of Sciences using the same manufacturing process as that used for the production of Pentavac SD. On days 1, 3, and 5 following each vaccination, the injection sites were evaluated by a veterinarian, blinded as to treatment, for redness, induration and swelling.

High-dose vaccination

Four animals were vaccinated once with a fourfold-concentrated wP vaccine (Pentavac SD), and four animals were vaccinated once with a fourfold-concentrated RRwP experimental vaccine. Each dose of fourfold-concentrated vaccine was generated by combining four doses of vaccine, centrifuging at 10,000 relative centrifugal force for 5 min, and resuspending the pelleted vaccine in 0.5 mL of the supernatant. On days 1, 2, 3, and 5 following each vaccination, the injection sites were evaluated by a veterinarian, blinded as to treatment. Relative scores were assigned for redness, induration, and swelling. In addition, the surface temperature at the injection site, and rectal temperature was recorded.

Infection and evaluation of animals

The 12 baboons vaccinated at 2, 4, and 6 months of age with the standard dose of the vaccine, and four age-matched, unvaccinated baboons were challenged with 108 to 109 CFUs of B. pertussis strain D420. To inoculate the baboons with B. pertussis, the baboons were anesthetized using 10 mg/kg ketamine intramuscularly. The animal’s pharynx was swabbed with a 2% lidocaine solution, and 1 mL of bacterial inoculum was delivered using a 2–3 mm diameter endotracheal tube. Additionally, 0.5 mL of inoculum was delivered in each nasopharyngeal cavity using a 22-gauge, 3.2-cm Teflon intravenous catheter (Abbott Laboratories, Chicago, IL). The animals were observed twice daily following challenge to assess health status. Whole blood and nasopharyngeal washes from baboons were collected under sedation twice weekly. Whole blood specimens were evaluated for the number of circulating white blood cells by complete blood count (CBC). Each nasopharyngeal cavity was flushed with 0.5 mL of phosphate-buffered saline (PBS), using a 22-gauge/3.2-cm intravenous catheter. The recovered nasopharyngeal washes from both nares were combined, and 100 µL of the recovered sample was diluted and plated on two Regan–Lowe plates. The number of B. pertussis CFUs/mL of nasopharyngeal wash was calculated after incubation at 37°C for 4–5 days.

Datalogger implantation and data analysis

Data loggers (Star-Oddi LTD, Gardabaer, Iceland) were implanted under anesthesia through a mid-scapular skin incision into a subcutaneous pocket created by blunt dissection. Sutures were placed subcutaneously to anchor and restrict migration of the device. The incision was closed in two layers with absorbable suture. The animals were monitored through full recovery from surgery.

The temperature data were recorded by the datalogger in 10-min intervals. Data analysis was performed using the pracma package (version 2.4.4) in R (version 4.4.1). Specifically, the trapz function was utilized to calculate the area under the curve (AUC) of the 18-h interval body temperature on day 1 post-vaccination recorded between 3:00 PM and 9:00 AM of the second day. The same analysis was also applied to the 3 days before the vaccination to establish the baseline. The 18-h interval between 3:00 PM and 9:00 AM was chosen to avoid the impact of the sedation performed in the morning of day 1 on body temperature.

PBMC restimulation

Blood was collected from baboons under sedation before challenge and following challenge to isolate PBMCs as described previously (23). Heat-killed B. pertussis (HKBp) was prepared by heat-inactivating B. pertussis D420 cells resuspended in PBS at OD600 0.9 for 30 min at 65°C. PBMCs were resuspended at 5 × 106 cells/mL in RPMI supplemented with 10% FBS, 1% 100× Gibco Antibiotic/Antimycotic Solution, 1% L-glutamine and 25 mM HEPES. The cells were incubated with or without HKBp (50 bacteria per PBMC) at 37°C. Supernatants were collected after 72 h.

Cytokine ELISA

Supernatants collected from PBMCs stimulated with or without HKBp were assessed for IL17a, IFNγ, and IL5 protein levels using an enzyme-linked immunosorbent assay (ELISA) (U-CyTech Biosciences, Utrecht, Netherlands) following the manufacturer’s instructions. In brief, 96-well plates were coated with anti-monkey pAb for specific cytokines at 4°C overnight. After 1% BSA in 1× PBS were used for 2 h to block nonspecific binding, samples and recombinant standards for the respective cytokine were added, and the ELISA plates were incubated for 2 h at 37°C. Bound cytokine was detected using cytokine-specific biotinylated mAb and streptividin-HRP, followed by Sure Blue TMB Microwell peroxidase (KPL, Gaithersburg, MD). Optical densities were read at 450 nm with Molecular Devices VERSA Max microplate reader and analyzed using SoftMax Pro software (version 7.1.2).

B. pertussis-specific IgG ELISAs

Levels of pertussis-specific serum IgG were measured as described previously (54). Nunc 96-well plates were coated with 0.2 µg/mL pertussis toxin (PT) (List Biologicals) or HKBp at OD600 0.9. Each plate included a standard curve using WHO international pertussis antiserum (National Institute for Biological Standards and Control), and IgG results were expressed as international units (IU)/mL or relative units (RU)/mL by comparing the results with the linear portion of the standard curve.

Intracellular staining

PBMCs isolated from blood post-challenge on days 7, 14, and 21 were stimulated with HKBp at 1 × 107 CFU/mL and cultured in the presence of co-stimulatory anti-CD28 and anti-CD49d antibodies both at a final concentration of 1 ug/mL for 16 h at 37°C in the presence of 5% CO2. Cellular protein transport was inhibited for another 5 h by adding GolgiPlug (BD Biosciences) at 1 uL/mL to capture intracellular cytokines. Fc block and surface stains (anti-CD45, anti-CD3, anti-CD4) were added and incubated on ice for 30 min in the dark. After washing with FACS buffer, the cells were fixed according to the BD Cytofix/Cytoperm Fixation/Permeabilization kit (BD Biosciences) instructions for 20 min at RT in the dark. The cells were then permeabilized by incubation with Perm/Wash Solution (BD Biosciences) while conducting intracellular staining with antibodies against IFNγ and IL17a for 30 min at RT in the dark. After staining, the cells were washed with Perm/Wash buffer and FACS buffer before acquisition. Acquisition was performed on a BD LSRFortessa X-20 flow cytometer (BD Biosciences). Data analyses were performed with FlowJo software (version 10).

Statistical analysis

Comparisons between groups were made using the nonparametric two-tailed unpaired Mann–Whitney U test or repeated measures two-way ANOVA. Prism 5.0 (GraphPad) and R 4.4.1 were used for the analysis. Data are presented as mean ± standard deviation (SD). P < 0.05 was considered statistically significant.

ACKNOWLEDGMENTS

This work was supported in part by the project National Institute of Virology and Bacteriology (Programme EXCELES, ID Project No. LX22NPO5103) - Funded by the European Union - Next Generation EU (P.S.) and by the Food and Drug Administration and by the National Institutes of Health Division of Microbiology and Infectious Disease through Interagency Agreement # AAI14017-001-00002.

Contributor Information

Tod J. Merkel, Email: tod.merkel@fda.hhs.gov.

Marcela F. Pasetti, University of Maryland School of Medicine, Baltimore, Maryland, USA

ETHICS APPROVAL

All animal procedures were performed in a facility accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International in accordance with protocols approved by the FDA’s Animal Care and Use Committee and the principles outlined in the Guide for the Care and Use of Laboratory Animals by the Institute for Laboratory Animal Resources, National Research Council.

SUPPLEMENTAL MATERIAL

The following material is available online at https://doi.org/10.1128/msphere.00647-24.

Supplemental Information. msphere.00647-24-s0001.docx.

Estimated coverage of whole-cell vaccines.

msphere.00647-24-s0001.docx (49.9KB, docx)
DOI: 10.1128/msphere.00647-24.SuF1

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Associated Data

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Supplementary Materials

Supplemental Information. msphere.00647-24-s0001.docx.

Estimated coverage of whole-cell vaccines.

msphere.00647-24-s0001.docx (49.9KB, docx)
DOI: 10.1128/msphere.00647-24.SuF1

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